Summary

The sense of balance depends on the intricate architecture of the inner
ear, which contains three semicircular canals used to detect motion of the
head in space. Changes in the shape of even one canal cause drastic behavioral
deficits, highlighting the need to understand the cellular and molecular
events that ensure perfect formation of this precise structure. During
development, the canals are sculpted from pouches that grow out of a simple
ball of epithelium, the otic vesicle. A key event is the fusion of two
opposing epithelial walls in the center of each pouch, thereby creating a
hollow canal. During the course of a gene trap mutagenesis screen to find new
genes required for canal morphogenesis, we discovered that the Ig superfamily
protein Lrig3 is necessary for lateral canal development. We show that this
phenotype is due to ectopic expression of the axon guidance molecule netrin 1
(Ntn1), which regulates basal lamina integrity in the fusion plate. Through a
series of genetic experiments, we show that mutually antagonistic interactions
between Lrig3 and Ntn1 create complementary expression
domains that define the future shape of the lateral canal. Remarkably, removal
of one copy of Ntn1 from Lrig3 mutants rescues both the
circling behavior and the canal malformation. Thus, the Lrig3/Ntn1
feedback loop dictates when and where basement membrane breakdown occurs
during canal development, revealing a new mechanism of complex tissue
morphogenesis.

INTRODUCTION

Precise spatiotemporal regulation of intercellular signaling is crucial for
molding tissues into three-dimensional structures during organogenesis. One of
the most striking examples of tissue morphogenesis is the development of the
three-dimensional architecture of the inner ear, which houses the sensory
organs for hearing and balance. Angular acceleration is detected by three
fluid-filled semicircular canals that are oriented with respect to the three
dimensions of space. Changes in head position result in fluid movement within
the canals, thereby activating specialized hair cells in the sensory epithelia
located at the base of each canal. Subsequently, vestibular ganglion neurons
convey signals from the sensory epithelium to the central nervous system. The
inner ear is a small, intricately shaped organ that does not tolerate even
subtle changes to its structure; indeed, even the slightest perturbations in
the structure of the vestibular canals can result in debilitating dizziness,
vertigo and abnormal posture in humans
(Sando et al., 2001;
Sando et al., 1984).

Morphogenesis of the inner ear is set in motion by early patterning events,
which result in the expression of key cell fate determinants within discrete
domains of a primordial structure. The inner ear is sculpted from a simple
ball of epithelium called the otic vesicle
(Fig. 1)
(Fekete, 1999). The
semicircular canals are derived from two outpocketings, the canal pouches,
which are specified by transcription factors such as Otx1 and Dlx5
(Merlo et al., 2002;
Morsli et al., 1999). A
crucial event in canal development is the formation of the fusion plate in the
center of the pouch. During this process, the basement membrane breaks down,
allowing signaling molecules to induce proliferation in the surrounding
mesenchyme and the two epithelial walls to come together
(Martin and Swanson, 1993;
Pirvola et al., 2004;
Salminen et al., 2000;
Streeter, 1907). At the same
time, fusion plate cells lose their columnar morphology and intercalate to
form a single layer of cells (Martin and
Swanson, 1993). Importantly, these changes in cell morphology and
basal lamina integrity occur only in the vicinity of the fusion plate. By
contrast, the epithelium in the perimeter of the pouch remains intact and will
eventually form the walls of the mature canal. Hence, the final shape of each
canal is determined by when and where fusion occurs.

Despite abundant evidence that Ntn1 is a powerful morphogen, little is
known about the pathways that restrict Ntn1 expression to highly
limited spatiotemporal domains in any developing system
(Kennedy, 2000). In addition
to its functions during development, Ntn1 is overexpressed in several human
cancers (Fitamant et al., 2008;
Link et al., 2007),
underscoring the importance of understanding how expression of Ntn1
is regulated. Here, we demonstrate that Ntn1 expression is controlled
by cross-repressive interactions with the Ig superfamily protein Lrig3 that
define the boundary between the fusing and non-fusing regions of the lateral
canal pouch. This novel feedback loop dictates when and where basement
membrane breakdown occurs, thereby ensuring that the inner ear acquires its
precise three-dimensional shape.

MATERIALS AND METHODS

Mice

The LST016 gene trap line has been previously reported
(Leighton et al., 2001;
Mitchell et al., 2001). The
mice have been maintained for over five generations on the C57Bl6/J
background. Genotyping was performed using the following primers: LST016F
(GAGGTGCCTGATGCTTAAGTTTCG); LST016R (TTCAACCTTGGCTTCCAATGTCCA); and GTR7
(CAAGTCTATCCTAGGGAAAGGGTC), which is specific to the
Lrig3LST016 gene trap vector pGT2TMPFS. The
Lrig3flox conditional allele was produced via homologous
recombination using a targeted construct with LoxP sites surrounding the
ATG-containing exon 1. Heterozygous mice were produced by germline
Cre-excision using a global deleter line
(Schwenk et al., 1995).
Genotyping was performed using the following primers: VEA303
(CGGAATTTCCTACAATCTCAGC); VEA304 (GTGCTCCTGGTGGCTCAGT); VEA305
(CCCCCTCCAATTTTAACAAA). The Ntn1 gene trap line has been previously
reported (Serafini et al.,
1996). The mice have been maintained for over five generations on
the C57Bl6/J background. Genotyping was performed using the following primers:
pGT1_8TM3021R (GTTGCACCACAGATGAAACG); pGTEn2_1723 (TCCCGAAAACCAAAGAAGAAG); and
pGT1_8TM1743 (GAACCCTAACAAAGAGGACAAG). The described genotyping screen allows
for the detection of the Ntn1-specific gene trap vector
pGT1.8TM. Heterozygous and mutant genotypes were confirmed by
evaluating the thickness of the ventral commissure in the neural tube
following βIII-Tubulin and Neurofilament immunostaining on cryosectioned
embryos. These two gene trap lines are annotated as
Lrig3+/- or Ntn1+/- for the
heterozygous condition and Lrig3-/- or
Ntn1-/- for the mutant condition. Unc5hb mutant
mice were maintained on a CD1 background and genotyped as described
(Lu et al., 2004). The
integrin α6 gene trap mice (Mitchell
et al., 2001) were maintained on a C57Bl6/J background and were
genotyped using the following primers: LST045F5 (GCCCAAATCCCTTGTGTATG);
LST045R1 (ACCCACAGCAACCTTTGTTC); and GTR3 (TCTA GGACAAGAGGGCGAGA). The mice
were maintained in accordance with institutional and NIH guidelines approved
by the IACUC at Harvard Medical School and the University of Virginia.

Patterning and morphogenesis of the inner ear. Diagrams of the
transformation of the otic vesicle into the mature structure of the inner ear.
Early in development (left), the axes of the otic vesicle are patterned, with
the presumptive vestibular system expressing Dlx5 and Hmx3
(red) and the developing cochlea expressing Otx2 (yellow). The
lateral pouch is defined by expression of Otx1 (blue stipple). A few
hours later, during morphogenesis, discrete regions in the dorsal and lateral
pouch begin to transcribe netrin 1 (blue, middle). These regions will
subsequently undergo fusion and disappear, leaving the epithelium in the
perimeter to form the walls of the mature canals (right). Motion is detected
by hair cells housed in swellings at the base of each canal called ampullae
(*). In all of the following figures, paintfilled inner ears are
shown looking down onto the lateral canal, with anterior towards the right,
whereas sections through the otic vesicle are in the transverse plane (as
indicated by a broken line), with dorsal upwards and lateral towards the
right.

Quantitative PCR

cDNA was made from total mouse E11.5 embryonic RNA using random primers.
Lrig3 was amplified as described
(Abraira et al., 2007) with
SYBR Green Supermix (BioRad) and primers flanking the Lrig3 exon3/4
boundary: VEA48 (GAACAACAATGAATTAGAGACCATTC) and VEA49 (AGGGTGGAAAGGCAGTTCTC).
Levels were normalized based on amplification of GAPDH from the same samples:
GAPDHe-F (CTCATGACCACAGTCCATGC) and GAPDHe-R (GCACGTCAGATCCACGAC). Loss of
Lrig3 message from Lrig3 null mice was confirmed by RT-PCR
amplification of E12.5 embryonic cDNA using primers VEA48 and VEA49.

Paintfilling

E12-E14.5 embryos were fixed overnight at 4°C with Bodian's Fix,
dehydrated overnight at room temperature with 100% ethanol, then cleared
overnight at room temperature with methyl salicylate. Heads were hemisected,
and white latex paint (Benjamin Moore) diluted to 0.025% in methyl salicylate
was injected into the cochlea with a pulled glass pipette and a Hamilton
syringe filled with glycerol.

In situ hybridization

Non-radioactive in situ hybridization was performed on 10-12 μm
cryosections using the following probes: Ntn1 (NM_008744),
Otx1 (NM_011023), Otx2 (NM_144841), Dlx5
(NM_010056) and Hmx3 (NM_008257). A detailed protocol is available at
http://neuro.med.harvard.edu/site/goodrichweb/.

X-gal and PLAP staining

Staining for β-galactosidase and alkaline phosphatase activity was
performed as described (Leighton et al.,
2001) except that 10-20 μm frozen sections were used, and the
tissue was fixed for 1 hour at 4°C.

Immunohistochemistry

E12 embryos were collected and fixed for 1-2 hours at 4°C in 4% PFA/PBS
and then dehydrated in 30% sucrose/PBS overnight at 4°C. Embryos were then
embedded in Neg50 (Richard-Allan Scientific). Cryosections (5-10 μm) were
blocked and permeabilized in 5% normal donkey serum + 2% BSA + 0.1% Triton
X-100 for 1 hour at room temperature. Primary antibodies were added into the
above block, without Triton X-100, overnight at 4°C at the following
concentrations: collagen IV (1:200, Abcam, ab6586); pan-mouse laminin (1:250,
Chemicon, AB2034); βIII-tubulin (1:1000, Covance, PRB-435P); and
neurofilament (1:500, DSHB, SH3). The following day, the sections were
incubated in secondary antibody (1:2000, Alexa Fluor488 or 568, Jackson
Immunoresearch) in block, without Triton X-100. All sections were
counterstained with DAPI (1:10,000).

Histology and electron microscopy

Plastic semi-thin (1 μm) and ultra-thin (90 nm) sections for light and
electron microscopy studies were obtained with help from the Harvard Medical
School Electron Microscopy Facility
(cellbio.med.harvard.edu/research_facilities).

RESULTS

Identification of Lrig3 as a new regulator of inner ear
morphogenesis

To gain insight into the molecules that drive inner ear morphogenesis, we
took advantage of the fact that even subtle changes in the structure of the
inner ear cause dramatic behavioral defects. Candidate genes were identified
by screening a large set of mice generated by gene trap technology
(Leighton et al., 2001;
Mitchell et al., 2001). Upon
insertion into the intron of a gene, the gene trap vector simultaneously
blocks transcription and reports the normal expression pattern of that gene
through a β-galactosidase reporter
(Fig. 2A). X-gal-stained
heterozygous embryos were examined to find genes expressed in restricted
domains of the otic vesicle prior to morphogenesis, and then homozygous
mutants for the best candidate genes were assessed for defects in hearing and
balance.

In this screen, we identified a strain of mice, LST016, withβ
-galactosidase activity in the lateral wall of the otic vesicle
(Fig. 2B,C), a region fated to
give rise to the lateral semicircular canal
(Fekete and Wu, 2002).
Homozygous mutants exhibit circling and head tossing behaviors, consistent
with the presence of an inner ear defect
(Fig. 2D). Visualization of the
three-dimensional structure of the vestibular apparatus revealed a fully
penetrant truncation of the lateral semicircular canal in homozygotes
(Fig. 2G-J). Histological
studies confirmed that the canal epithelium is missing as early as E13 (data
not shown). The lateral ampulla is unaffected, and the sensory epithelium is
properly innervated, as determined by myosin VIIa and neurofilament
immunostaining (Fig. 2I,J; data
not shown). Although the reporter is also active in the developing cochlea
(see Fig. S1H in the supplementary material), the mice develop normal hearing
as assessed by auditory brainstem response (ABR) assays (see Fig. S2 in the
supplementary material). All mutants also display craniofacial deformities and
a dramatically shortened snout (Fig.
2E,F).

Lrig3 mutant mice exhibit circling behavior owing to a
truncation of the lateral semicircular canal. (A) Diagrams of two
independent alleles of Lrig3 illustrating insertion of the gene trap
vector in LST016 mice (left) and the introduction of LoxP sites on
either side of the ATG-bearing exon in the conditional
Lrig3flox allele (right). (B,C) X-gal
detection of the Lrig3-β-geo fusion protein in an E10.5 Lrig3
heterozygous embryo (B) and in sections through the otic vesicle (C) in the
plane indicated (broken line, B). β-Galactosidase activity is high in
somitic mesoderm, the branchial arches and the limb buds. In the developing
inner ear, transcription of Lrig3 is enriched in the lateral otic
epithelium by E10.5 (bracket). Scale bar: 50 μm. (D) A single
Lrig3 mutant mouse photographed in three points of its circling
trajectory. (E,F) Lrig3 homozygotes (F) have shortened
snouts compared with heterozygotes (E). (G-J) Paintfilled inner ears of
E14 Lrig3+/- (G,I) and Lrig3-/- (H,J)
embryos. Low magnification views of the entire inner ear (G,H) reveal a
truncation of the lateral semicircular canal (arrowhead, H). Other structures
appear normal in size and shape. High magnification views of the vestibular
apparatus confirm truncation of the lateral canal (LC) but not the anterior
(AC) or posterior (PC) canals. The lateral ampullae (asterisks) are
unaffected, with no change in the number or distribution of
MyosinVIIa-positive hair cells in the lateral cristae (insets). Scale bar: 50μ
m. Dorsal is upwards; posterior is towards the left.

LST016 mice carry an insertion of the pGT2TMPFS gene trap
vector in the third intron of the Lrig3 gene
(Fig. 2A), causing a truncation
of the wild-type transcript and fusion of the β-galactosidase reporter
protein to Lrig3 at amino acid 126
(Mitchell et al., 2001). Lrig3
is one of three members in a family of single-pass transmembrane proteins
containing 15 leucine-rich repeats (LRR) and three immunoglobulin (Ig) domains
in the extracellular domain and intracellular tails that vary in length and
composition (Guo et al., 2004;
Hedman and Henriksson, 2007).
Quantitative RT-PCR of homozygous Lrig3 tissue (n=4 embryos)
detected only 3.05±1.59% of the wild-type transcript, indicating that
the phenotype is severely hypomorphic. To confirm that the phenotype is due to
a loss of Lrig3, a null allele was made by flanking the ATG-bearing
exon of Lrig3 with LoxP sites
(Fig. 2A). After germline
Cre-mediated excision, no Lrig3 messenger RNA remained (data not
shown). Null mutant mice exhibit the same lateral canal and craniofacial
defects evident in the gene trap allele (data not shown), demonstrating that
the phenotypes reflect a complete loss of Lrig3 function.

Truncation of the lateral semicircular canal in Lrig3
homozygotes is due to early and ectopic fusion

The semicircular canals develop from two epithelial outpocketings called
canal pouches, with the anterior and posterior canals arising from the dorsal
pouch and the lateral canal forming from the lateral pouch
(Fig. 1). Early patterning
events that define the axes of the otic vesicle result in restricted
expression of transcription factors that specify the two pouches. Because
Lrig3 expression is restricted to the lateral pouch during these
early patterning stages, it seemed possible that Lrig3 acts in a signaling
pathway that ensures restricted expression of transcription factors required
for specification of the lateral canal. To test this, we examined the
expression patterns of Otx1, Otx2, Hmx3 and Dlx5,
transcription factors required for normal development of the vestibular system
(Merlo et al., 2002;
Morsli et al., 1999;
Wang et al., 1998). However,
Lrig3 mutant embryos exhibited no obvious changes in early gene
expression, indicating that the lateral pouch is specified in the right place
and at the right time (see Fig. S3 in supplementary material).

As canal patterning was unaffected, we asked whether subsequent
morphogenesis events proceed normally in Lrig3 mutant embryos. We
visualized pouch outgrowth and fusion by paintfilling inner ears between E11.5
and E12.5 (Fig. 3). The precise
stage of canal development was determined by evaluating the extent of fusion
in the anterior and posterior canals, which develop earlier than the lateral
canal (Martin and Swanson,
1993). In control embryos, the lateral pouch grows out at E12 and
fusion begins at E12.5 (Fig.
3A-C). No changes in the size or shape of the early canal pouch
occur in E12 Lrig3 mutant embryos, consistent with correct patterning
of the otic vesicle (Fig. 3D).
However, fusion in the lateral pouch begins several hours earlier than normal
and over a larger area, extending to the perimeter of the lateral pouch
epithelium (Fig. 3E). The
lateral canal is truncated by E12.5, when fusion is just beginning in
wild-type littermates (Fig.
3F). Thus, fusion is expanded and accelerated in Lrig3
mutants.

Lrig3 acts in the non-fusing epithelium to prevent premature and ectopic
fusion. (A-F) Dorsal views of paintfilled
Lrig3+/- (A-C) and Lrig3-/- (D-F)
inner ears collected at 6-hour intervals from E12 to E12.5. Anterior is
towards the right. In mutants, early outgrowth is normal (D). However, fusion
initiates too early (E) and occurs over a larger area (asterisk) than in
control embryos (B,C), resulting in truncation by E12.5 (F).
(G-I′) Adjacent sections of Lrig3 heterozygotes through
the lateral pouch at the level indicated (broken line, A) were processed forβ
-galactosidase histochemistry to reveal Lrig3-βgeo activity (G-I)
or for in situ hybridization with a probe to Ntn1
(G′-I′). Lrig3 is transcribed throughout the lateral
pouch prior to fusion (G). Levels become reduced (H) in the nascent fusion
plate (arrowhead) just as Ntn1 expression initiates here (H′).
Lrig3 expression is further diminished (I) as transcription of
Ntn1 expands (I′). Scale bar: 50 μm.

Although fusion occurs prematurely in Lrig3 mutants, fusion is
arrested in Ntn1 mutants, raising the possibility that these two
genes cooperate to determine the timing and extent of fusion. To gain more
insight into the relationship between Lrig3 and Ntn1 during
canal morphogenesis, we compared their expression patterns on adjacent
sections from E12 to E12.5. We found that Lrig3 expression is
enriched in the lateral pouch epithelium during the canal pouch outgrowth
stage, when Ntn1 is not yet expressed
(Fig. 3G,G′). Then, just
before fusion begins, Lrig3 expression becomes reduced in the center
of the pouch (Fig. 3H),
concomitant with the initiation of Ntn1 transcription in the nascent
fusion plate (Fig. 3H′).
As fusion progresses, the domain of Ntn1 transcription expands
(Fig. 3I′), whereas
Lrig3 is downregulated in Ntn1-positive cells
(Fig. 3I) but is maintained in
the surrounding, non-fusing epithelium. After the canal is fully formed,
Lrig3 and Ntn1 continue to be expressed in non-overlapping
domains of the canal epithelium (data not shown). Hence, consistent with their
opposing activities during canal development, Lrig3 and Ntn1
are expressed in complementary domains of the lateral pouch.

These results suggest that Lrig3 is required in the non-fusing
epithelium to prevent fusion from occurring by balancing the activity of
Ntn1 in the fusion plate. Malformation of the semicircular canals in
Ntn1 mutant mice is preceded by a failure in basement membrane
breakdown (Salminen et al.,
2000). Therefore, we asked whether the early and ectopic fusion
event in Lrig3 mutant mice is also accompanied by changes in the
integrity of the basal lamina that normally separates the epithelium from the
surrounding mesenchyme. We found that dramatic changes occur in the basal
lamina of Lrig3 mutant embryos. In control embryos, laminin and
collagen networks are intact prior to fusion plate formation, as shown by
immunostaining and electron microscopy analysis
(Fig. 4A-E). By contrast, in
Lrig3 mutants, the basal lamina is missing or disrupted throughout
the lateral pouch, including regions where fusion normally never occurs
(Fig. 4F-J). Moreover, the otic
epithelium throughout the lateral pouch is abnormally thin, such that even
cells in the perimeter of the pouch resemble fusion plate cells
(Fig. 4A′,F′).
Neither caspase 3 immunostaining (see Fig. S4 in the supplementary material)
nor electron microscopy analysis (data not shown) revealed an increase in
apoptosis, suggesting that these morphological changes are not associated with
cell death. Thus, Lrig3 inhibits basement membrane breakdown in the non-fusing
epithelium.

Ntn1-dependent basal lamina breakdown does not require known
receptors

To understand the molecular basis of the basement membrane phenotype, we
explored the possibility that Lrig3 modulates Ntn1 activity by regulating one
of its known receptors. This hypothesis is supported by the facts that
Lrig3 is expressed complementary to Ntn1 and that Lrig
proteins regulate degradation of many different transmembrane receptors
(Hedman and Henriksson, 2007).
Ntn1 is best known as an axon guidance molecule, which signals through the Ig
superfamily of receptors DCC (deleted in colorectal cancer) and neogenin 1, as
well as the Unc5 family of receptors (Unc5Ha-d)
(Moore et al., 2007). Outside
of the nervous system, Ntn1 signals through neogenin
(Srinivasan et al., 2003),
Unc5hb (Lu et al., 2004) and
integrin α3/6 receptors (Yebra et
al., 2003) to regulate cell adhesion, migration and other aspects
of tissue morphogenesis.

To identify relevant receptors, we asked whether any of the known Ntn1
receptors is required for inner ear morphogenesis. In situ hybridization
screens by our laboratory and others revealed Unc5hb, neogenin and
integrin α3/6 as potential candidates
(Matilainen et al., 2007).
However, although Unc5hb is expressed together with Lrig3 in
non-fusing epithelium, the inner ear forms normally in Unc5hb mutant
mice (see Fig. S5 in the supplementary material). Moreover, none of the known
Ntn1 receptors that are expressed in the developing otic epithelium (neogenin,
integrin α3/6) or surrounding mesenchyme (Unc5hc) are required
for canal morphogenesis (see Fig. S5 in the supplementary material)
(Matilainen et al., 2007).

Interestingly, the pro-angiogenic activities of Ntn1 have been proposed to
be independent of known receptors, suggesting an alternative binding partner
in this system (Wilson et al.,
2006). Although Lrig3 is a cell-surface protein, Lrig3 does not
appear to be the missing receptor, as tagged versions of Lrig3 and Ntn1 do not
colocalize in cultured cell lines (see Fig. S6 in the supplementary material).
Moreover, equal amounts of Ntn1 are secreted from cells in the presence and
absence of Lrig3 (see Fig. S6 in the supplementary material). Thus, Ntn1
appears to act through a non-canonical pathway in the inner ear to control
basal lamina integrity, either via a novel receptor or a receptor-independent
mechanism.

The basement membrane undergoes early and ectopic breakdown in the inner
ear of Lrig3 mutant mice. (A,F) Transverse plastic
sections through inner ears of E12 Lrig3+/- (A) and
Lrig3-/- (F) littermates, with magnified views
of the boxed areas (A',F'). Dorsal is upwards; lateral is towards the right.
In controls, epithelial cells in the fusion plate intercalate to form a single
layer of cells (A′), but in mutants, this region is expanded (brackets,
F′). Scale bar: 50 μm. (B,C,G,H)
Immunofluorescent detection of collagen IV (B,G) and all laminins (C,H) in E12
Lrig3+/- (B,C) and Lrig3-/-
embryos (G,H) sectioned in the transverse plane. In homozygotes, the basal
lamina is disturbed by breaks in the laminin network and ectopic accumulation
of collagen IV (arrowheads, G,H). Scale bar: 50 μm.
(D,E,I,J) Electron micrographs of the regions
indicated in C and H. The basement membrane is continuous in heterozygotes
(arrowheads, D,E) but is absent (asterisks, I) or severely disrupted
(asterisk, J) in homozygotes. Scale bar: 500 nm.

Lrig3 and Ntn1 participate in cross-repressive
interactions that define the fusing and non-fusing domains of the lateral
pouch. (A,E) In situ hybridization of Ntn1 on
transverse sections through E12 Lrig3+/- (A) and
Lrig3-/- (E) embryos. In Lrig3 mutants,
Ntn1 expression is expanded to fill the lateral pouch (outlined).
(B,F) β-galactosidase histochemistry of E12
Lrig3+/- (B) and Lrig3-/- (F)
littermates. As previously demonstrated
(Fig. 3), Lrig3-βgeo
levels are reduced in fusion plate cells (arrowhead, B) compared with the
surrounding epithelium. However, in Lrig3 mutants (F) reporter
activity is present at high levels throughout the pouch. (C,G)
Lrig3 and Ntn1 mutant mice were generated with two different
gene trap vectors, so only Lrig3LST016 mice carry a
placental alkaline phosphatase (PLAP) reporter. Hence, PLAP histochemistry
reveals Lrig3 transcription in
Ntn1+/+;Lrig3+/- (B) and
Ntn1-/-; Lrig3+/- embryos (G) at E12.5. Likeβ
-geo, PLAP staining of Lrig3 heterozygotes (C) is absent from
the fusion plate at E12.5 (arrowhead). By contrast, Lrig3
transcription is sustained in the fusion plate of age-matched Ntn1
homozygotes (G). Note that these embryos are 12 hours older than those in
A,B,E,F. (D,H) β-Galactosidase histochemistry of E12
Ntn1+/- (D) and Ntn1-/- (H)
littermates. Ntn1-βgeo is active in the fusion plate (arrowhead) in
heterozygotes (D), consistent with in situ hybridization results (see
Fig. 3). However, no activity
is detected in the lateral pouch of Ntn1 homozygotes (H).
Ntn1-βgeo expression is unchanged in the dorsal pouch (asterisks). Scale
bar: 50 μm.

Lrig3 and Ntn1 participate in cross-repressive
interactions that determine the timing and location of fusion

As Lrig3 does not appear to act through the Ntn1 pathway, we considered
alternative explanations for why basement membrane breakdown is expanded in
Lrig3 mutants. Based on their complementary expression patterns and
opposing activities, a simple idea is that the main function of Lrig3 is to
restrict expression of Ntn1 to the fusion plate. As hypothesized, we
found that Ntn1 expression is expanded to encompass the entire
lateral pouch epithelium in Lrig3 mutants
(Fig. 5A,E). As Lrig3 appears
to regulate Ntn1 expression at the transcriptional level, we asked
whether the transcriptional downregulation of Lrig3 in the wild-type
fusion plate (Fig. 3) is
similarly a result of Ntn1 activity. To do this, we took advantage of the fact
that a placental alkaline phosphatase (PLAP) reporter gene is present in the
gene trap vector used to generate Lrig3 mutants
(Fig. 2A), but not in
Ntn1 gene trap mutants (Serafini
et al., 1996). As seen by β-galactosidase histochemistry
(Fig. 3I), PLAP expression is
reduced in the fusion plate in Lrig3 heterozygous embryos
(Fig. 5C). By contrast, in
Ntn1 mutants, PLAP expression persists throughout the lateral pouch
(Fig. 5G). Surprisingly, we
also observed sustained expression of Lrig3 itself in Lrig3
mutants, indicating that Lrig3 is required for its own downregulation
(Fig. 5B,F). Therefore, we
asked whether Ntn1 transcription is also autoregulated. Indeed,
Ntn1-βgeo expression is lost from the lateral pouch in Ntn1
mutants (Fig. 5D,H). Ntn1
autoregulation appears to be limited to the lateral pouch, as no changes in
expression occur in the dorsal pouch. Together, these experiments show that
mutually antagonistic interactions between Lrig3 and Ntn1
restrict Ntn1 expression to fusion plate cells and Lrig3
expression to non-fusing epithelium. This feedback loop seems to be uniquely
important for lateral canal development, suggesting that new mechanisms
operate in this most recently evolved semicircular canal.

The Lrig3 mutant lateral canal truncation is rescued by removal
of one copy of Ntn1. (A-F) Paintfilled E14.5 inner ears
from transheterozygous intercross littermates. Canals (arrowheads) are normal
in wild-type (A) and transheterozygous (B) embryos, whereas the lateral canal
is truncated in Lrig3 homozygotes (C). The truncation is fully
rescued when one wild-type copy of Ntn1 is removed from
Lrig3 homozygotes (D). Fusion does not occur in Ntn1 mutants
(E) or in Ntn1;Lrig3 double mutants (F). (G) Table summarizing
the proportion of ears with normal, truncated or unfused semicircular canals
for each genotype in offspring from Ntn1+/-;
Lrig3+/- intercrosses. The rescued population is highlighted
in green. Note that the Ntn1 phenotype is not influenced by the
absence of Lrig3.

Loss of one copy of Ntn1 is sufficient to rescue inner ear
defects in Lrig3 mutants

The presence of cross-repressive interactions between Lrig3 and
Ntn1 suggests that a slight reduction in Ntn1 levels might
be sufficient to compensate for the loss of Lrig3. To test this idea,
we reduced the dose of Ntn1 in Lrig3 mutant mice by
intercrossing Ntn1+/-; Lrig3+/-
transheterozygotes and collecting E14.5 embryos (n=81) for
paintfilling (Fig. 6).
Wild-type (n=4/4) and transheterozygous (n=23/23) embryos
developed normal canals (Fig.
6A,B,G), whereas littermates homozygous for Lrig3 but
wild type for Ntn1 exhibited lateral canal truncations, as expected
(n=11/12 ears) (Fig.
6C,G). By contrast, no truncations occurred in Lrig3
homozygotes that were also heterozygous for Ntn1 (n=12/12
ears) (Fig. 6D,G), indicating
that the Lrig3 phenotype is caused by a failure to properly regulate
Ntn1 levels in fusion plate cells. Consistent with the complete
rescue, adult Lrig3-/-;Ntn1+/- mice did not
display circling behavior (n=16). As the size and shape of the canal
pouch is similar in Ntn1 (Fig.
6E,G) (n=7) and Ntn1;Lrig3 double mutant embryos
(Fig. 6F,G) (n=4),
Lrig3 probably acts upstream of Ntn1.

We conclude that Lrig3 plays a pivotal role in lateral canal
morphogenesis by restricting Ntn1 expression to the fusion plate.
Cross-repressive interactions between Lrig3 and Ntn1
coordinate the timing and location of fusion, thereby determining the shape of
the lateral canal.

DISCUSSION

A central challenge in developmental biology is understanding how signaling
pathways cooperate to sculpt tissues with complex three-dimensional shapes.
Here, we describe the presence of a novel feedback loop that restricts the
expression of two genes with opposing functions to discrete domains of the
otic vesicle during inner ear morphogenesis. Because the pattern of
Ntn1 expression determines when and where fusion occurs, the
spatiotemporal regulation of Ntn1 through cross-repressive
interactions with Lrig3 ensures perfect morphogenesis of the
elaborate structure of the inner ear.

Historically, much emphasis has been placed on the identification of
determinants that define domains within the anlage of a developing structure.
More recently, however, it has become clear that an additional level of
control is needed to restrict the spatiotemporal activities of each factor. A
common solution to this problem is the production of a feedback-induced
antagonist that dampens the activity of a signaling pathway after activation,
as is the case for Sprouty and Sef proteins in the FGF pathway
(Shim et al., 2005;
Tsang and Dawid, 2004).
Signaling activity is also modulated by the basal lamina, which can limit
dispersal of the protein and its ability to bind to its receptor
(Relan and Schuger, 1999). The
Lrig3/Ntn1 feedback loop incorporates both of these features: the
induction of an antagonist, Lrig3, which controls the timing and
extent of Ntn1-dependent basal lamina breakdown.

The Ntn1/Lrig3 feedback loop uncovered by this analysis is a novel
mechanism for modulating expression of Ntn1 during development. In
addition to the developing canals, Lrig3 and Ntn1 are also
expressed in many other regions of the embryo, including the cochlea, neural
tube and somites (see Fig. S1 in the supplementary material). Although no
alterations of these tissues are obvious in Lrig3 mutant mice (data
not shown), this may be due to compensation by the close family member
Lrig1. Indeed, our expression studies indicate that the only two
places where Lrig1 and Lrig3 do not overlap are the lateral
canal pouch and the branchial arches, consistent with the lateral canal
truncation and craniofacial abnormalities evident in Lrig3 mutant
mice. Conversely, psoriasis is the only salient defect reported in
Lrig1 mutant mice (Suzuki et al.,
2002). Hence, Lrig1; Lrig3 double mutants may exhibit
additional phenotypes related to misregulation of Ntn1 and may reveal
a general mechanism for Ntn1 regulation.

Proposed model for the Lrig3/Ntn1 feedback loop. (A) A
diagrammatic view of the lateral pouch during canal morphogenesis.
Cross-repressive interactions between Lrig3 and Ntn1 define
the boundary between fusing (blue) and non-fusing (red) regions of the otic
epithelium. When the regulatory loop is interrupted by loss of Lrig3,
fusion is expanded, whereas in Ntn1 mutants, fusion does not occur.
Interactions between Lrig3 and Ntn1 ensure that these two
genes become confined to distinct domains of the lateral pouch. (B) We
propose the following model for the Lrig3/Ntn1 feedback
loop. Lrig3 is present throughout the canal pouch before fusion and inhibits
activity of a receptor tyrosine kinase (RTK) signaling pathway (1).
Subsequently, fusion is initiated by an unknown fusion plate signal, which we
hypothesize acts through the RTK pathway by inhibiting Lrig3 (2), resulting in
transcription of Ntn1 (3). Lrig3 initially continues to be
transcribed, as expected for a feedback-induced antagonist. However, the
increased levels of Ntn1 eventually inhibit Lrig3 expression, either
by inhibiting Lrig3 or by potentiating activity of the fusion plate signal.
For example, Ntn1 protein may augment activity of the RTK pathway by promoting
basal lamina breakdown (4).

Based on the current understanding of this poorly characterized family of
proteins, it is likely that Lrig3 represses Ntn1 transcription by
regulating activity of a receptor tyrosine kinase signaling pathway. The
best-studied family member, Lrig1, is induced by EGF signaling and antagonizes
downstream signaling events by causing degradation of all four ErbB receptors
(Gur et al., 2004;
Laederich et al., 2004). Lrig1
has also been implicated as a negative regulator of Met and Ret receptor
tyrosine kinases (RTKs) (Ledda et al.,
2008; Shattuck et al.,
2007), raising the possibility that Lrig proteins serve as general
antagonists of RTKs. Consistent with this idea, Lrig3 can bind to the FGF
receptor tyrosine kinase receptor in vitro and inhibits FGF signaling in the
developing neural crest in Xenopus
(Zhao et al., 2008). This
function may be conserved in mice, as Lrig3 morphant tadpoles exhibit
craniofacial defects similar to what is observed in Lrig3 mutant
mice. As FGF signaling plays a prominent role in canal morphogenesis
(Chang et al., 2004;
Pauley et al., 2003;
Pirvola et al., 2004), the
Lrig3 inner ear phenotype is also likely to be caused by aberrant FGF
activity. However, although FGF ligands and receptors have been implicated,
the downstream signaling events in the fusion plate remain elusive, with no
known target genes and multiple feedback-induced antagonists that are not
required for canal development (Abraira et
al., 2007; Shim et al.,
2005). Hence, until we have a better understanding of how FGF
signaling acts specifically in the fusion plate, it will be difficult to
determine whether and how Lrig3 influences FGF activity.

Together with what is known about Lrig function, the simplest
interpretation of our results is that Lrig3 titrates activity of a receptor
tyrosine kinase signaling pathway that normally induces expression of
Ntn1, as well as Lrig3 itself
(Fig. 7). Prior to fusion,
Lrig3 is expressed throughout the lateral pouch, where it inhibits
RTK activity and prevents fusion from beginning
(Fig. 7, step 1). Subsequently,
we hypothesize the presence of a fusion plate inducing signal, which overcomes
Lrig3-mediated inhibition (Fig.
7, step 2) to activate the RTK pathway and allow expression of
Ntn1 (Fig. 7, step 3).
Ntn1, in turn, enhances activity of this same pathway, most probably by
promoting breakdown of the basal lamina
(Fig. 7, step 4). Hence, in
Lrig3 mutants, increased RTK signaling results in early and expanded
Ntn1 expression, as well as persistent Lrig3 expression
(Fig. 7A). Conversely, in
Ntn1 mutants, RTK signaling occurs at low levels, both because of the
presence of Lrig3 and the failure to potentiate the pathway through
Ntn1-mediated breakdown of the basal lamina. Owing to the low level of RTK
signaling, canal development arrests at the canal pouch stage, such that
expression of Ntn1 is lost and Lrig3 is never reduced
(Fig. 7A).

A key feature of this model is the fact that Lrig3 is present prior to any
of these events, but is also crucial for the subsequent emergence of mutually
exclusive domains of Lrig3 and Ntn1 expression. As Lrig3
activity is required for the feedback loop to function properly, the initial
Lrig3 expression also fails to be downregulated in Lrig3
mutants. Hence, the subsequent interactions between Lrig3 and
Ntn1 serve to simultaneously reduce Lrig3 transcription and
increase Ntn1 only in the regions that receive the inducing
signal.

The proposed model fits both with the results reported here as well as the
known activities of Lrig3 and Ntn1, but also raises several questions for
future consideration. Unfortunately, the ability of Lrig proteins to bind to
widely divergent members of the receptor tyrosine kinase family in vitro will
make it difficult to pinpoint a single binding partner in vivo, especially in
a structure as small as the lateral pouch. The best candidate is the FGF
receptor, not only because Lrig3 is known to bind to and inhibit FGF receptor,
but also because of the known importance of FGF signaling during inner ear
development. In the developing vestibular system, FGF signaling cooperates
with the BMP pathway to define sensory and non-sensory domains of the inner
ear (Chang et al., 2004;
Pauley et al., 2003) and is
subsequently required for proliferation of the periotic mesenchyme
(Pirvola et al., 2004).
Moreover, mesenchymal proliferation is reduced in Ntn1 mutants,
consistent with the idea that Ntn1-dependent breakdown of the basal lamina
promotes the ability of the FGF ligand to act during canal morphogenesis.
Indeed, it is well established that FGF signaling levels are modified by
interactions with the basement membrane during other types of tissue
morphogenesis (Lonai, 2003;
Patel et al., 2007). Although
it remains unclear how Ntn1 mediates its effects, the extent of basement
membrane breakdown correlates strongly with the amount of Ntn1:
breakdown does not occur in Ntn1 mutants, is expanded in the presence
of ectopic Ntn1 in Lrig3 mutants, and proceeds normally in
rescued embryos that have only one copy of Ntn1. Additional
experiments will be needed to understand the specific function of Ntn1 in the
basement membrane, as well as how Ntn1-induced changes influence the activity
of FGF or other signaling ligands in the extracellular matrix.

Because modest perturbations in the structure of the inner ear cause severe
behavioral deficits, our genetic studies were able to reveal the consequences
of slight changes in signaling activity that may be undetectable by in vitro
methods. Indeed, lateral canal truncations also occur in BMP4
heterozygotes, emphasizing the unusual sensitivity of the developing inner ear
to modest changes in signaling levels
(Chang et al., 2008).
Similarly, in humans, the lateral canal is the most common site of inner ear
anomalies (Sando et al., 2001;
Sando et al., 1984),
emphasizing the importance of identifying the molecular players that make this
canal unusually susceptible to developmental insults. Hence, the
Lrig3/Ntn1 feedback loop may provide an additional safeguard
for tissues whose function depends crucially on the perfect morphogenesis of
complex structures.

Supplementary material

Acknowledgments

We thank X. Lu for providing Unc5hb mutant tissue, D. Goodenough
for insights into the EM results, N. Hyun and N. Pogue for genotyping
assistance, and R. Segal, N. Andrews, X. Lu, M. Scott and members of the
Goodrich laboratory for advice on the project and comments on the manuscript.
We are also grateful to S. Walker and J. Brugge for assistance with the acinar
cultures. This work was supported by grants R01 DC7195 (L.V.G.) and F31
DC008450 (V.E.A.) from the N.I.H./N.I.D.C.D., and by funding from the C.I.H.R.
(J.S.), the Smith Family New Investigators Program (L.V.G.) and the Mathers
Charitable Foundation (L.V.G.).

Similar articles

Other journals from The Company of Biologists

Although sad to be saying goodbye to Ottoline Leyser and Geraldine Seydoux as they leave our Editorial Board, we are very pleased to introduce two new editors, Yka Helariutta and Susan Strome. Olivier Pourquié has also announced that he will also be stepping down next year, and so we invite the Development community to help us make the right choice for our next Editor in Chief.

“It's good advice that you collaborate – you need to have so many aspects to what you are doing and you can't possibly be an expert in every one”

Jenny Nichols, winner of the 2017 BSDB Cheryll Tickle Medal, talks about the importance of collaboration throughout her career investigating pluripotency in the mammalian embryo and what playing a musical instrument has in common with research.

Organised by Paola Arlotta, Ali Brivanlou, Olivier Pourquié and Jason Spence, the third meeting in our highly successful series of events focussing on human developmental biology will be held at Wotton House in Surrey, UK, on 23 - 26 September 2018. Mark it on your calendar now!

The authors of a Research article on how PLCζ-null mice can still be fertile even in the absence of the Ca2+ oscillations that induce egg activation chat to the Node about their research, looking positively on bad luck and bringing science to life for children.

Dorit Hockman from the University of Oxford studies the development and evolution of the neural crest by investigating its development in lampreys. A Travelling Fellowship from Development gave Dorit the opportunity to visit Marianne Bronner’s laboratory at the California Institute of Technology over the lamprey breeding season, where she learned how to track neural crest cells as they migrated into the branchial arches. Read her story here.

Where could your research take you? The deadline for the current round of applications for a Development Travelling Fellowship is 31 August. Find out more here.